宫颈癌相关新癌基因hWAPL的原核表达和免疫原性分析

目的:构建hWAPL原核表达载体,诱导hWAPL蛋白表达并制备其多克隆抗体。方法:RT-PCR技术扩增hWAPL cDNA片段,连接到pMD18-T载体,测序正确后亚克隆入原核表达载体pET28a并转化BL21菌株,用SDS-PAGE电泳鉴定重组菌的诱导表达情况,制备hWAPL蛋白并免疫BALB/c小鼠,ELISA法测定其抗体效价,Western blot鉴定表达hWAPL蛋白的免疫原性。结果:①经PCR、双酶切鉴定和测序,证实hWAPL正确插入pMD18-T载体,序列正确。②经IPTG诱导的pET28a-hWAPL重组菌表达出相对分子质量(Mr)约为25000的蛋白,与预期蛋白Mr相符。③免疫小鼠后测得的平均抗体效价为1∶3200。④Western blot结果显示Mr约25000处有反应蛋白条带。结论:pET28a载体能够表达hWAPL蛋白,表达蛋白具有良好的免疫原性,其免疫小鼠后获得的多克隆抗体具有高效价和特异性。

Prokaryotic expression and immunogenicity of hWAPL, a norel oncogene related to cervical cancer

AIM: To construct the prokaryotic expression plasmid of hWAPL, induce the expression of the protein and prepare polyclonal antibody. METHODS: The hWAPL cDNA was amplified by RT-PCR from HeLa cells derived RNA and then cloned to pMD18-T according to A-T. The DNA fragment was isolated and linked to prokaryotic expression vector pET28a. The recombinant protein was induced by IPTG and identified by SDS-PAGE. Then it was injected into mice to prepare polyclonal antibody. The immunogenicity and specificity of the recombinant protein were identified by ELISA and Western blot. RESULTS: The sequencing, PCR and endonucleases digestion results showed that the hWAPL fragment was correctly inserted into pMD18-T and pET28a vectors. SDS-PAGE showed 25,000 fusion hWAPL protein was expressed in BL21 cells. The titer of polyclonal antibody was 1:3 200 by indirect ELISA. The 25,000 fusion hWAPL protein was detected by SDS-PAGE and Western blot. CONCLUSION: The hWAPL protein can be expressed by prokaryotic vector pET28a. Furthermore, the expression protein can be used as immunogen to generate antibodies with specificity.